Cells in vivo reside in an organized three-dimensional environment as part of tissue and organ structures; loss of this tissue organization is a hallmark of cancer (Wodarz, et al., Nature Cell Biology 9:1016-1024 (2007); Lee, et al., J. Cell Sci. 121:1141-1150 (2008); Morrison, et al., Nature 441:1068-1074 (2006)). For nearly a century, progress in cancer research and discovery of anti-cancer agents have been fueled by investigations performed on cells cultured outside the living organism (ex vivo) (Ebeling, J. Exp. Med. 17:273-285 (1913); Carrel, et al., J. Exp. Med. 13:387-U34 (1911); Carrel, et al., J. Exp. Med. 13:571-575 (1911); Leighton, Cancer Res. 17:929-941 (1957); Paul, Cancer Res. 22:431-& (1962)). The majority of these studies have been performed with cells cultured on two dimensional surfaces. Morphological and functional differences of cells cultured in these conditions and cells in vivo have been widely recognized, and three-dimensional cell growth substrates have been shown to present a more physiologically relevant model of in vivo cell environment (Yamada, et al., Cell 130:601-610 (2007); Nelson, et al., Annu. Rev. Cell Dev. Biol. 22:287-309 (2006); Huang, et al., Nature cell biology 1:E131-E138 (1999); Schmeichel, et al., J. Cell Sci. 116, 2377-2388 (2003)). Importantly, cells cultured on two-dimensional substrates often do not respond to soluble factors that influence cells in three-dimensional environments (Emerman, et al., In Vitro-Journal of the Tissue Culture Association 13:316-328 (1977); Emerman, et al., Proc. Natl. Acad. Sci. U.S.A. 74:4466-4470 (1977); Cukierman, et al., Science 294:1708-1712 (2001); Bissell, et al., Differentiation 70:537-546 (2002); Weaver, et al., J. Cell Biol. 137:231-245 (1997)). Yet, to date, the majority of drug discovery processes start from small-molecule screening in two-dimensional culture-based assays. Failures of the identified compounds in animal and human trials drive the cost of drug discovery to >$1 billion per new compound (Griffith, et al., Nat. Rev. Mol. Cell Biol. 7:211-224 (2006)). High-throughput assays based on three-dimensional cultures can allow assessment of drug efficiency and toxicity at the very first step of the drug discovery process. Integration of the three-dimensional cell culture into every aspect of basic and applied cancer research will advance the discovery of new therapeutics for cancer treatment.
Differences in cell responses in three-dimensional vs. two-dimensional environment originate from differences in cell polarity, cellwide distribution of the substrate adhesion sites and responses of cells to mechanical properties of the matrix (Yamada, et al. 2007; Huang, et al., 1999). On the molecular level, these events are regulated by the cross-talk between integrin signaling pathways and those of the receptor-tyrosine kinases. Properties like chemical composition of the matrix, nano- and microscale distribution of the integrin ligands (Cukierman, et al., 2001; Chen, et al., Science 276:1425-1428 (1997)), as well as mechanical property of the matrix (Engler, et al., Cell 126:677-689 (2006); Pelham, et al., Proc. Natl. Acad. Sci. U.S.A. 94:13661-13665 (1997); Yeung, et al., Cell Motil. Cytoskeleton 60:24-34 (2005)), can influence these pathways and modulate cell behavior. Furthermore, delivery of oxygen and nutrients to cells in gel-like matrix is driven by diffusion; hence, physical dimensions of the matrix also play a role in three-dimensional cell culture. Due to diffusion limitations, proliferation of cells in three-dimensional matrices ex vivo is often limited to a depth of less than a few hundred microns. Therefore, the size, composition, and mechanical properties of the matrix must be carefully controlled in three-dimensional culture.
After decades of side-by-side development of two-dimensional and three-dimensional culture, the simplicity of two-dimensional culture approach makes it a dominant technology for ex vivo investigation of cells. The need to control multiple chemical and physical properties of the matrix makes three-dimensional culture of cells more labor intensive and less reproducible.